Defects in motor neuron (MN) function or survival result in severe human pathologies, such as amyotrophic lateral sclerosis and spinal muscular atrophy, with distinct MN subtypes differing in their susceptibility to disease. There is currently no effective treatment for these disorders, in part due to a lack of understanding of the molecular mechanisms that allow distinct MN subtypes to acquire and maintain their function-defining properties. The continuous expression, from development through adulthood, of subtype-specific terminal identity genes (e.g., genes coding for ion channels, neurotransmitter receptors, neuropeptides, signaling molecules) defines the unique, functional features of a given MN subtype throughout life. How these genes are induced and maintained is poorly understood. This proposal uses a novel approach to specifically focus on the transcriptional regulation of MN subtype-specific terminal identity genes. The goal of this proposal is to uncover conserved, gene regulatory mechanisms that establish during development, and maintain throughout life, the expression of MN subtype-specific terminal features. To this end, this proposal combines the strengths of two model organisms: the nematode Caenorhabditis elegans and mouse Mus musculus. By studying how C. elegans ventral nerve cord (VNC) MNs acquire their subtype-specific features, we discovered a gene regulatory mechanism that involves the intersectional activity of highly conserved transcription factors. We found that the transcription factor UNC-3 induces and maintains the expression of terminal identity genes in all VNC MN subtypes. However, UNC-3 does not act alone. It requires co-factors in the form of Hox proteins that act synergistically with UNC-3 to activate expression of terminal identity genes in distinct MN subtypes along the anterior-posterior axis of the VNC. We observed Hox expression in developing and adult MNs, suggesting that Hox proteins, similar to UNC-3, not only induce, but also maintain expression of terminal identity genes. The unpublished data in this application indicate that Hox proteins, in mice, also control expression of terminal identity genes in spinal MNs, suggesting evolutionary conservation of our C. elegans findings. This proposal aims to uncover the function of Hox proteins in adult C. elegans MNs (Aim 1), decipher the gene regulatory mechanisms downstream of Hox in C. elegans MNs (Aim 2), and test the hypothesis that a mouse Hox protein (Hoxc8), similar to its C. elegans orthologs, is required to induce and maintain expression of terminal identity genes in MNs of the brachial spinal cord (Aim 3). Completion of the proposed activities will advance our understanding of how distinct MN subtypes become and remain functional, which may provide new insights into the etiology, diagnosis, and treatment of MN disorders.